Project Summary: The gram-negative bacterial outer membrane provides an efficient permeability barrier for both hydrophilic and hydrophobic compounds, and, as a result, bacteria have transport systems to facilitate the passage of water-soluble nutrients and other molecules across the outer membrane. However, little is known about the transport of hydrophobic substrates across the outer membrane. The FadL family is the only outer membrane protein family known to function as substrate-specific transporters of hydrophobic compounds. While FadL is required for long-chain fatty acid uptake, FadL homologues are present in gram-negative bacteria that degrade hydrophobic aromatic hydrocarbons, such as the BTEX (benzene, toluene, ethylbenzene, xylene) compounds. The aims of this research are (1) to examine the structural basis for the transport of aromatic hydrocarbons via outer membrane proteins and (2) to characterize the substrate specificity of FadL homologues for hydrophobic compounds. Membrane protein purification and X-ray crystallography will be the primary techniques used to elucidate the structural mechanisms for hydrophobic compound transport. To address the substrate specificity of FadL homologues for hydrophobic compounds, including fatty acids and aromatic hydrocarbons, in vivo and in vitro substrate binding and transport assays will be developed. Although the proposed research specifically focuses on the characterization of transport proteins involved in aromatic hydrocarbon uptake, a broader outcome will be a more comprehensive appreciation of how hydrophobic compounds are transported across the gram-negative outer membrane. Relevance: Aromatic hydrocarbons, particularly the BTEX compounds, are toxic, mutagenic and carcinogenic and pose a significant hazard to human health and to the environment. The continued use of hydrocarbons in industrial manufacturing processes has resulted in considerable environmental contamination. Biodegradation of aromatic hydrocarbons by microorganisms offers a viable option for remediating contaminated sites, and understanding the mechanisms for microbial hydrocarbon uptake will advance our knowledge of biodegradation processes in the environment.